Bottom Line:
Anticancer drug therapy activates both molecular cell death and autophagy pathways.Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death.Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.

ABSTRACTAnticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.

f1: Sublethal concentrations of anticancer drugs induce multinucleated cells and autophagy.(a) Morphological analysis. Jurkat T cells were cultured in the presence and absence of the indicated anticancer drugs for 48 h. Drug-treated cells were classified as normal, apoptotic or abnormal, with enlarged and irregular nuclei, with multipolar mitoses or multinucleated. Cultures treated with etoposide (0.25 μM), cisplatin (3 μM), taxol (4 nM) and nocodazole (10 nM) exhibited high levels of cells with abnormal nuclei. None of the treated populations showed a significant fraction of apoptosis (see also Supplementary Fig. S1). Values are means±s.d. for three independent experiments. Right: representative examples of these morphological categories are shown. Scale bar, 10 μM. Similar abnormalities were also seen after treating NB4 and MDA-MA-231 cells with etoposide or cisplatin (Supplementary Fig. S2). (b) Immunoblotting. Jurkat T cells were treated as indicated and ATG5 (monomeric and ATG12-conjugated), ATG12 (conjugated with ATG5), LC3 (18 and 16 kDa), Beclin 1 and GAPDH detected. Chloroquine (CQ) was employed to block lysosomal proteases. Each immunoblot is representative of at least three independent experiments. The same assays were performed using NB4 and MDA-MA-231 cells (Supplementary Fig. S2). Moreover, as a marker for autophagy onset, increased LC3-II formation following etoposide or cisplatin treatment was also documented using fluorescence microscopy (Supplementary Fig. S2). Full-length immunoblots are provided in Supplementary Fig. S8.

Mentions:
To investigate the role of autophagy in anticancer drug treatment, we decided to study the effects of different drugs at sublethal concentrations as determined for six antimitotic and DNA-damaging drugs in multiple experiments (the procedure is illustrated in Supplementary Fig. S1). Jurkat T cells were examined over the first 4 days after treatment with these six different drugs, each at the defined sublethal concentration, and classified morphologically as normal, apoptotic or abnormal. The latter case comprised enlarged cells with enlarged, irregular nuclei, cells exhibiting abnormal, multipolar mitoses and multinucleated cells (Fig. 1a). The apoptotic fraction was relatively modest at 2–6% (Fig. 1a). Cells treated with etoposide, cisplatin, taxol or nocodazole, however, showed 40–80% abnormal morphology, exhibiting enlarged, irregular nuclei, multipolar mitoses, or multiple nuclei, characteristic of mitotic catastrophe5. Mitotic catastrophe is defined in international consensus as an oncosuppressive phenomenon occurring during or after defective mitosis leading to death or senescence.6 On the other hand, inefficient mitotic catastrophe with mitotic slippage can lead to the emergence of genetically unstable, tumorigenic, aneuploid cell population567.

f1: Sublethal concentrations of anticancer drugs induce multinucleated cells and autophagy.(a) Morphological analysis. Jurkat T cells were cultured in the presence and absence of the indicated anticancer drugs for 48 h. Drug-treated cells were classified as normal, apoptotic or abnormal, with enlarged and irregular nuclei, with multipolar mitoses or multinucleated. Cultures treated with etoposide (0.25 μM), cisplatin (3 μM), taxol (4 nM) and nocodazole (10 nM) exhibited high levels of cells with abnormal nuclei. None of the treated populations showed a significant fraction of apoptosis (see also Supplementary Fig. S1). Values are means±s.d. for three independent experiments. Right: representative examples of these morphological categories are shown. Scale bar, 10 μM. Similar abnormalities were also seen after treating NB4 and MDA-MA-231 cells with etoposide or cisplatin (Supplementary Fig. S2). (b) Immunoblotting. Jurkat T cells were treated as indicated and ATG5 (monomeric and ATG12-conjugated), ATG12 (conjugated with ATG5), LC3 (18 and 16 kDa), Beclin 1 and GAPDH detected. Chloroquine (CQ) was employed to block lysosomal proteases. Each immunoblot is representative of at least three independent experiments. The same assays were performed using NB4 and MDA-MA-231 cells (Supplementary Fig. S2). Moreover, as a marker for autophagy onset, increased LC3-II formation following etoposide or cisplatin treatment was also documented using fluorescence microscopy (Supplementary Fig. S2). Full-length immunoblots are provided in Supplementary Fig. S8.

Mentions:
To investigate the role of autophagy in anticancer drug treatment, we decided to study the effects of different drugs at sublethal concentrations as determined for six antimitotic and DNA-damaging drugs in multiple experiments (the procedure is illustrated in Supplementary Fig. S1). Jurkat T cells were examined over the first 4 days after treatment with these six different drugs, each at the defined sublethal concentration, and classified morphologically as normal, apoptotic or abnormal. The latter case comprised enlarged cells with enlarged, irregular nuclei, cells exhibiting abnormal, multipolar mitoses and multinucleated cells (Fig. 1a). The apoptotic fraction was relatively modest at 2–6% (Fig. 1a). Cells treated with etoposide, cisplatin, taxol or nocodazole, however, showed 40–80% abnormal morphology, exhibiting enlarged, irregular nuclei, multipolar mitoses, or multiple nuclei, characteristic of mitotic catastrophe5. Mitotic catastrophe is defined in international consensus as an oncosuppressive phenomenon occurring during or after defective mitosis leading to death or senescence.6 On the other hand, inefficient mitotic catastrophe with mitotic slippage can lead to the emergence of genetically unstable, tumorigenic, aneuploid cell population567.

Bottom Line:
Anticancer drug therapy activates both molecular cell death and autophagy pathways.Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death.Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.

ABSTRACTAnticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.